Flow cytometric measurement of cytoplasmic free calcium in human peripheral blood T lymphocytes with fluo-3, a new fluorescent calcium indicator

J Immunol Methods. 1990 Mar 9;127(2):197-205. doi: 10.1016/0022-1759(90)90069-8.


Cytoplasmic free calcium [( Ca2+]i) is a key intracellular messenger in many cell types. We have used fluo-3, a recently developed calcium probe, to study [Ca2+]i in resting and stimulated human peripheral blood T lymphocytes. The spectral properties of fluo-3 permit analysis of [Ca2+]i in flow cytometers with a 488 nm argon laser excitation source and fluorescein filter settings. The data obtained with fluo-3 are both qualitatively and quantitatively in good agreement with the data in the literature. After stimulation of T lymphocytes with the mitogens phytohaemagglutinin, concanavalin A and with OKT3, and anti-CD3 monoclonal antibody, a biphasic [Ca2+]i response was observed, with an early EGTA-insensitive [Ca2+]i rise, followed by an EGTA-sensitive sustained [Ca2+]i plateau. Non-mitogenic monoclonal antibodies directed against the CD5, CD28 and CD7 T cell surface antigens elicited [Ca2+]i rises only when crosslinked on the cell surface with goat anti-mouse IgG. Conversion of fluorescence data into absolute [Ca2+]i values by means of a non-disruptive calibration procedure, yielded a [Ca2+]i of 107 +/- 18 nM (mean +/- SD, n = 13) in resting T lymphocytes. Time-dependent loss of cellular dye content limits the precision of the calibration procedure in experiments of longer duration. We conclude that fluo-3 promisingly extends the potential application field of flow cytometers with 488 nm argon lasers to [Ca2+]i studies in T lymphocytes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds*
  • Antibodies, Monoclonal / immunology
  • Antigens, Surface / immunology
  • Calcium / analysis*
  • Calibration
  • Egtazic Acid / pharmacology
  • Flow Cytometry*
  • Fluorescence
  • Humans
  • Lymphocyte Activation
  • T-Lymphocytes / analysis*
  • Xanthenes*


  • Aniline Compounds
  • Antibodies, Monoclonal
  • Antigens, Surface
  • Xanthenes
  • Fluo-3
  • Egtazic Acid
  • Calcium